Metabolites and small molecules are important for the activity and regulation of many eukaryotic proteins. Although a large number of natural compounds that bind proteins have been identified through the analysis of individual interactions, a systematic screen for cellular compounds that bind proteins has not been performed. We have established approaches using high throughput protein purification and mass spectrometry as well as protein microarrays to identify protein-small molecule interactions in yeast. Using the mass spectrometry approach, we have found that several yeast proteins involved in the ergosterol biosynthetic pathway and many protein kinases bind hydrophobic molecules. Of these, at least one compound, ergosterol, affects protein kinase activity directly. Building upon these findings, we propose to systematically identify small molecules that bind yeast proteins on a proteome scale. We will use large scale protein purification and mass spectrometry to determine the suite of both hydrophobic and hydrophillic molecules that binds yeast proteins, initially focusing our attention on phosphatases and transcription factors then extending this analysis to as much of the proteome as possible. The identity of the bound compounds will be determined by comparison with properties of other known compounds. We will use an independent and parallel approach to globally deduce in vitro binding partners for 100 metabolites by probing yeast proteome arrays with small molecules. The information found in this study will be used to identify new biochemical activities, reconstruct metabolic networks and explore novel aspects and basic principles of regulatory networks. The information from this study will be made available to the general scientific community through an online accessible database and deposition in public databases. Overall this study will provide the first global analysis of small molecule-protein interactions in eukaryotes, and is expected to provide novel insights into protein function and regulation. PUBLIC HEALTH RELEVANCE: Metabolites and small molecules participate in a wide variety of biochemical and regulatory functions. They serve as metabolic components, cofactors for enzymes, forms of energy for biochemical reactions and regulators of protein function and gene expression. In spite of their importance, systematic approaches for analyzing these interactions have not been performed. Such information is expected to be valuable not only for elucidating the biochemical activities and regulation of individual proteins, but also for assembling and understanding regulatory networks and connections between biological pathways. We plan to utilize novel approaches that we have developed to determine the metabolites and small molecules that bind to important proteins that regulate cellular events using yeast as a model system. This study will then be extended to find the molecules that bind most yeast proteins. The information is expected to yield novel insights into the functions of many proteins, how they are regulated, and aid in understanding how biochemical pathways are regulated and coordinated. This project ultimately has high significance for understanding and improving human health. By understanding how proteins and pathways are regulated by metabolites we can attempt to manipulate metabolite levels to help control human diseases that have defects in pathways that are controlled or affected by metabolites. Metabolite levels are particularly amenable for therapies since many drugs already exist that regulate metabolite levels and levels can often be controlled through dietary restriction or supplement. Thus, this study may have direct application to the improvement of human health in the future.